Depletion of<i>SLC4A11</i>Causes Cell Death by Apoptosis in an Immortalized Human Corneal Endothelial Cell Line

Liu, Jun; Seet, Li‐Fong; Koh, Li Wei; Venkatraman, Anandalakshmi; Venkataraman, Divya; Mohan, Rajiv R.; Prætorius, Jeppe; Bonanno, Joseph A.; Aung, Tin; Vithana, Eranga N.

doi:10.1167/iovs.11-8724

Cited by 38 publications

(35 citation statements)

References 30 publications

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“…20,22 Moreover, it was recently shown that the knock-down of SLC4A11 causes a reduction of cellular growth and proliferation in HeLa cells and that a depletion of the SLC4A11 gene alone may already lead to apoptosis induction in CECs. 25,26 Prostaglandin-endoperoxide synthase 2 (PTGS2, COX2) was further evaluated in the early-onset FECD mouse model and in human late-onset FECD. PTGS2 was chosen due to its 5-fold endothelial transcriptional up-regulation in mutant animals, the previously established corneal endothelial expression of cyclooxygenases and its modulation capability with readily available drugs like COX2 inhibitors.…”

Section: Discussionmentioning

confidence: 99%

Endothelial Cell Whole Genome Expression Analysis in a Mouse Model of Early-Onset Fuchs' Endothelial Corneal Dystrophy

Matthaei

Hu²,

Meng³

et al. 2013

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. To investigate the endothelial gene expression profile in a Col8a2 Q455K mutant knock-in mouse model of earlyonset Fuchs' endothelial corneal dystrophy (FECD) and identify potential targets that can be correlated to human late-onset FECD.METHODS. Diseased or normal endothelial phenotypes were verified in 12-month-old homozygous Col8a2 Q455K/Q455K mutant and wild-type mice by clinical confocal microscopy. An endothelial whole genome expression profile was generated by microarray-based analysis. Result validation was performed by real-time PCR. Endothelial COX2 and JUN expression was further studied in human late-onset FECD compared to normal samples.RESULTS. Microarray analysis demonstrated endothelial expression of 24,538 genes (162 up-regulated and 172 down-regulated targets) and identified affected gene ontology terms including Response to Stress, Protein Metabolic Process, Protein Folding, Regulation of Apoptosis, and Transporter Activity. Real-time PCR assessment confirmed increased Cox2 (P ¼ 0.001) and Jun mRNA (P ¼ 0.03) levels in Col8a2 Q455K/Q455K mutant compared to wild-type mice. In human FECD samples, real-time PCR demonstrated a statistically significant increase in COX2 mRNA (P < 0.0001) and JUN mRNA (P ¼ 0.002) and tissue microarray analysis showed increased endothelial COX2 (P ¼ 0.02) and JUN protein (P ¼ 0.04).CONCLUSIONS. The present study provides the first endothelial whole genome expression analysis in an animal model of FECD and represents a useful resource for future studies of the disease. In particular endothelial COX2 up-regulation warrants further investigation of its role in FECD. (Invest Ophthalmol Vis Sci.

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Section: Discussionmentioning

confidence: 99%

Endothelial Cell Whole Genome Expression Analysis in a Mouse Model of Early-Onset Fuchs' Endothelial Corneal Dystrophy

Matthaei

Hu²,

Meng³

et al. 2013

Invest. Ophthalmol. Vis. Sci.

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“…In CHED2 and FECD patients, there is a gradual reduction in endothelial cell number in addition to loss of function [Klintworth, 2009;Liu et al, 2012]. Accumulation of ER-retained proteins, in some cases, leads to apoptotic cell death [Xu et al, 2005].…”

Section: Expression Of Slc4a11 Mutants Does Not Induce Apoptosis or Cmentioning

confidence: 99%

Corneal Dystrophy-Causing SLC4A11 Mutants: Suitability for Folding-Correction Therapy

Loganathan

Casey

2014

Human Mutation

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SLC4A11 mutations cause some cases of the corneal endothelial dystrophies, congenital hereditary endothelial corneal dystrophy type 2 (CHED2), Harboyan syndrome (HS), and Fuchs endothelial corneal dystrophy (FECD). SLC4A11 protein was recently identified as facilitating water flux across membranes. SLC4A11 point mutations usually cause SLC4A11 misfolding and retention in the endoplasmic reticulum (ER). We set about to test the feasibility of rescuing misfolded SLC4A11 protein to the plasma membrane as a therapeutic approach. Using a transfected HEK293 cell model, we measured functional activity present in cells expressing SLC4A11 variants in combinations representing the state found in CHED2 carriers, affected CHED2, FECD individuals, and unaffected individuals. These cells manifest respectively about 60%, 5%, and 25% of the water flux activity, relative to the unaffected (WT alone). ER-retained CHED2 mutant SLC4A11 protein could be rescued to the plasma membrane, where it conferred 25%-30% of WT water flux level. Further, some ER-retained CHED2 mutants expressed at 30°C supported increased water flux compared with 37°C cultures. Caspase activation and cell vitality assays revealed that expression of SLC4A11 mutants in HEK293 cells does not induce cell death. We conclude that therapeutics able to increase cell surface localization of ER-retained SLC4A11 mutants hold promise to treat CHED2 and FECD patients.

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“…41 Mutant SLC4A11 does not induce apoptosis in HEK293 cells on its own. 27 One report found that HEK293 cells transfected with mutant SLC4A11 decrease expression of antioxidant proteins, rendering the cells more susceptible to oxidative stress.…”

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confidence: 94%

“…38 Human SLC4A11 was originally reported to be a Na þ -coupled borate transporter, 37 but other groups have not been able to replicate this finding. 39,40 Instead, SLC4A11 has been found to facilitate Na þ /OH À transport, 41 NH 3 /H þ cotransport, 42 and electrogenic H þ (OH À ) permeation. 43 Human SLC4A11 mediates water movement when expressed in Xenopus laevis oocytes and HEK293 cells, 44 which makes it the first identified water transporter that is not a member of the major intrinsic protein family.…”

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confidence: 99%

High Throughput Assay Identifies Glafenine as a Corrector for the Folding Defect in Corneal Dystrophy–Causing Mutants of SLC4A11

Chiu

Mandziuk

Loganathan

et al. 2015

Invest. Ophthalmol. Vis. Sci.

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Citation: Chiu AM, Mandziuk JJ, Loganathan SK, Alka K, Casey JR. High throughput assay identifies glafenine as a corrector for the folding defect in corneal dystrophy-causing mutants of SLC4A11. Invest Ophthalmol Vis Sci. 2015;56:7739-7753. DOI:10.1167/ iovs.15-17802 PURPOSE. Protein misfolding, causing retention of nascent protein in the endoplasmic reticulum (ER), is the most common molecular phenotype for disease alleles of membrane proteins. Strategies are needed to identify therapeutics able to correct such folding/trafficking defects. Mutations of SLC4A11, a plasma membrane transport protein of the human corneal endothelial cell layer, cause cases of congenital hereditary endothelial dystrophy, Harboyan syndrome, and Fuchs' endothelial corneal dystrophy. Most SLC4A11 mutations induce SLC4A11 misfolding and retention in the ER.METHODS. An assay amenable to high-throughput screening was developed to quantify SLC4A11 at the plasma membrane, enabling a search for potential traffic-correcting small molecules. The assay was validated by comparing cell surface abundance of SLC4A11 mutants measured in the assay to observations from confocal immunofluorescence and values from cell surface biotinylation. Functionality of mutant proteins was assessed, using a confocal microscopic green fluorescent protein (GFP) water flux assay where relative rates of cell swelling are compared. RESULTS.A small-scale screen revealed that the nonsteroidal anti-inflammatory drugs (NSAIDs), glafenine, ibuprofen, and acetylsalicylic acid dissolved in 0.2% dimethyl sulfoxide (DMSO), partially rescued the trafficking defect in some SLC4A11 mutants, expressed in HEK293 cells. These SLC4A11 mutants retained functional activity when rescued to the plasma membrane by glafenine treatment. Glafenine was effective with an EC 50 of 1.5 6 0.7 lM.CONCLUSIONS. These data suggest that glafenine, and perhaps other NSAIDs, hold potential as therapeutics for misfolded membrane proteins, like SLC4A11. The high throughput approach described here can be modified to identify correctors of other misfolded plasma membrane proteins that cause eye disease.

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Depletion ofSLC4A11Causes Cell Death by Apoptosis in an Immortalized Human Corneal Endothelial Cell Line

Cited by 38 publications

References 30 publications

Endothelial Cell Whole Genome Expression Analysis in a Mouse Model of Early-Onset Fuchs' Endothelial Corneal Dystrophy

Endothelial Cell Whole Genome Expression Analysis in a Mouse Model of Early-Onset Fuchs' Endothelial Corneal Dystrophy

Corneal Dystrophy-Causing SLC4A11 Mutants: Suitability for Folding-Correction Therapy

High Throughput Assay Identifies Glafenine as a Corrector for the Folding Defect in Corneal Dystrophy–Causing Mutants of SLC4A11

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